Technical Field
The present invention belongs to the field of biomedicine and relates to antibodies or functional fragments thereof that bind specifically to PD-1 with high affinity. The invention provides nucleic acid molecules encoding the antibodies or the fragments thereof according to the present invention, expression vectors and host cells for expressing the antibodies or the functional fragments thereof according to the present invention, as well as methods for producing the antibodies or the functional fragments thereof according to the present invention. The present invention also provides immunoconjugates and pharmaceutical compositions comprising the antibodies or the functional fragments thereof according to the present invention. The present invention additionally provides methods for treating a plurality of diseases (comprising cancers, infectious diseases and inflammatory diseases) by using the antibodies or the functional fragments thereof disclosed herein.
Description of Related Art
The programmed cell death 1 protein, PD-1, is a member of CD28 family and an immunosuppressive receptor expressed on the surfaces of the activated T cells and B cells (Yao, Zhu et al., Advances in targeting cell surface signaling molecules for immune modulation. Nat Rev Drug Discov, 2013, 12(2): 130-146). This receptor can bind to its ligands PD-L1 and PD-L2 to effectively reduce the immune response involving T cells. Tumor cells can escape the immune surveillance inside the body via high expression of PD-L1 (Okazaki and Honjo, PD-1 and PD-1 ligands: from discovery to clinical application. International Immunology, 2007, 19(7): 813-824 2007). The interaction between PD1 and PD-L1 can be blocked to significantly improve the tumor-killing activity of the CD8+ cytotoxic T cells.
PD-1 is primarily expressed on the surface of CD4+ T cells, CD8+ T cells, NKT cells, B cells, and the activated monocytes. The expression of PD-1 is primarily induced by the signals of T cell receptor (TCR) or B cell receptor (BCR). TNF can enhance the expression of PD-1 on the surfaces of these cells (Francisco, Sage et al., The PD-1 pathway in tolerance and autoimmunity. Immunol Rev, 2010, 236: 219-242). Human PD-1 is encoded by the gene Pdcd1, which is located on 2q37.3 and is 9.6 kb. It comprises five exons and four introns and its upstream comprises a promoter of 663 bp. The molecular structure of PD-1 comprises extracellular region, trans-membrane region and intracellular region. The amino acid sequences in the extracellular region has 24% homology with CTLA-4 and 28% homology with CD28. Its gene has primarily seven single-nucleotide polymorphic sites. The extracellular region comprises one structural domain of immuno-globulin variable IgV. The intracellular region comprises two signal transduction motifs based on tyrosine—ITIM (immunoreceptor tyrosine-based inhibitory motif) and ITSM (immunoreceptor tyrosine-based conversion motif). Once T cells are activated, PD-1 will associate with tyrosine phosphatase SHP2 primarily via the ITSM motif to cause the de-phosphorylation of the effector molecules including CD3ζ PKCθ and ZAP70, etc.
There are two PD-1 ligands: PD-L1 and PD-L2. PD-L1 is also referred to as B7H1 or CD274 and PD-L2 is referred to as B7DC or CD273. The PD-L gene is located on the locus of 9p24.2 of human chromosome with a size of 42 kb. These ligands have 21-27% homology in amino acid sequence and structural similarity with B7-1, B7-2 and ICOSL. The PD-1 ligands all comprise one structural domain of immuno-globulin-like variable region, one constant-region-like structural domain, one trans-membrane region, and one short cytoplast tail. The cytoplast tail of PD-L1 is more conservative than that of PD-L2. PD-L1 and PD-L2 are expressed on different cell populations (Shimauchi, Kabashima et al., Augmented expression of programmed death-1 in both neoplastic and non-neoplastic CD4+ T cells in adult T cell leukemia/lymphoma. Int J Cancer, 2007, 121(12): 2585-2590). These cells include non-hematopoietic tissue and a variety of tumor types. PD-L1 is mainly expressed on T cells, B cells, dendritic cells, macrophages, mesenchymal stem cells and mast cells derived from bone marrow. PD-L1 is also expressed on the cells not derived from bone marrow, such as vascular endothelial cells, epithelial cells, skeletal muscle cells, hepatocytes, renal tubular epithelial cells, islet cells, brain astrocytes, and various types of non-lymphoid tumors such as melanoma, liver cancer, stomach cancer, renal cell carcinoma, as well as expressed on the cells at the immunologically privileged sites such as placenta, eyes. It has been suggested that PD-L1 can be extensive to some degree in regulating the auto-reactive T cells, B cells and immune tolerance and can play a role in response of peripheral tissue T cells and B cells. Nevertheless, the PD-L2 has very limited expressed region and exists only in macrophages and dendritic cells. PD-L1 is believed to play a role mainly in the immune presentation.
PD-1 and PD-L1 interact with each other to regulate and control the activation of T cells, which has been validated by much in tumors and viral infections. PD-L1 is expressed on the surfaces of various tumor cells which include lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, renal carcinoma, stomach cancer, esophageal cancer, oral squamous cell cancer, and head/neck cancer. There are large amount of CD8+ T cells expressing PD-L1 found around these cancers. The clinical statistics reveal that the high level of expression of PD-L1 on cancer cells is related to the poor prognosis of cancer patients (Okazaki and Honjo 2007. Supra.).
Many chronic and acute viruses also escape the body's immune surveillance via the signals of PD-1 and PD-L1. For example, the expression level of PD-1 in HIV-infected patients is closely related to the degree of depletion of T cells and can be used as one of the markers of AIDS progression (Trabattoni, Saresella et al., B7-H1 is up-regulated in HIV infection and is a novel surrogate marker of disease progression. Blood, 2003, 101(7): 2514-2520). It is the same case for the patients diagnosed with chronic hepatitis B (Evans, Riva et al., Programmed death 1 expression during antiviral treatment of chronic hepatitis B: Impact of hepatitis B e-antigen seroconversion, Hepatology, 2008, 48(3): 759-769). The animal tests revealed that the mice whose PD-1 gene has been knocked out can control virus infection better than the normal mice; and hepatitis can be induced if the HBV-specific T cells are transferred into the HBV transgenic animals.